Triple negative breast cancer (TNBC), which includes tumors lacking estrogen, progesterone, and HER-2 receptor proteins, represents 15-20% of all breast cancer diagnoses in the US, and is one of the most challenging cancers for developing an effective therapy post tumor resection. Even with conventional radiation and chemotherapy regimens, patients have poor prognosis, experiencing early, frequent relapses in comparison to other breast cancers. New therapies are critically needed. We propose to develop and test an immunotherapy to treat metastatic TNBC using an approach that is personalized, easy to accomplish, and not requiring establishment of cell lines or gene transfer. The defining principle of this proprietary cancer immunotherapy technology is the incorporation of glycolipid anchored forms of immunostimulatory molecules (GPI-ISMs), such as B7-1 and IL-12, onto tumor membrane vesicles (TMV) derived from surgically removed patient tissue. We expect that these membrane attached immunostimulatory molecules will simultaneously deliver tumor antigens and activate immune cells to promote an effective anti-tumor immune response. The novelty of the proposed vaccine lies not in the use of immunostimulatory molecules but in the way they are employed. The protein transfer approach is substantially different from previous studies that used gene transfer techniques or secretory cytokines to develop cancer vaccines. The major advantage is that the immunotherapy preparation can be accomplished within just a few days. Additionally, tumor membrane preparations can be stored as frozen aliquots and biobanked for booster injections. These factors will significantly reduce the cost of immunotherapy production and treatment. In this Phase I application, we will use this protein transfer technique for membrane-based breast cancer immunotherapy and test its efficacy in inhibiting triple negative breast cancer metastasis in a murine model. Specifically, we will: 1) manufacture GPI-ISMs, optimize TMV processing and protein transfer, and evaluate efficacy using a murine metastatic TNBC model, 2) (A) Determine whether the membrane-based immunotherapy prevents/reduces metastasis of TNBC. (B) Investigate whether co-administration of anti-immunosuppressive agents augments efficacy of the membrane- based TNBC immunotherapy. Completion of the proposed SBIR Phase I feasibility studies will demonstrate the efficacy of membrane-based immunotherapy in inhibiting the metastatic growth of triple negative breast cancers. This will pave the way for Phase II studies such as toxicity and standardization of GMP/GLP conditions for large-scale production of purified human GPI-molecules, which will help us advance the vaccine product to human clinical trials.